JP2017014201A - Vehicle for delivering compound to mucous membrane and related composition, method and system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel agent useful for the treatment of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis.SOLUTION: A pharmaceutical composition has outer membrane vesicles formed by a lipid membrane enclosing an aqueous environment and comprising Polysaccharide A (PSA), the lipid membrane obtained from a cell membrane of Bacteroides fragilis. The PSA is preferably low molecular weight PSA (L-PSA).SELECTED DRAWING: Figure 1B

Description

  The Government of the United States has been awarded the grant number awarded by the National Institutes of Health. According to DK078938, we have some rights in this invention.

  The present disclosure relates to media, compositions, methods, and systems for delivering compounds to the mucosa. In particular, the present disclosure relates to a medium for delivering a compound to the mucosa of the gastrointestinal tract.

  Efficient delivery of the desired compound or substance to the mucosa continues to be challenging, particularly in delivering to the gastrointestinal tract and in particular to the human gastrointestinal tract.

  The human gastrointestinal tract possesses a large number of microorganisms (known as the gut microbiota) that have a wide range of effects on the progression and function of the immune system. Among the myriad bacterial species present in the mammalian digestive tract, the Bacteroides gate is the most abundant Gram-negative bacterial gate.

  It is particularly interesting to identify microorganisms and compounds involved in immune regulation. For example, Bacteroides fragilis is a microorganism that has been shown to have immunomodulatory properties and coexists with humans. In particular, the immunomodulatory properties of Bacteroides fragilis are associated with the production of polysaccharide A (PSA) by bacteria.

  Gram-negative bacteria have developed a complex mechanism for producing bacterial products, the type III secretion apparatus (T3SS), T4SS, and T6SS, which are bacterial effectors after direct contact with host cells. Making complex surface organs that change the position of the. Another secretory strategy is outer membrane media (OMVs) that are released from the surface of Gram-negative bacteria and send a set of cargo molecules to distant target cells. Many (but not all) gram-negative bacteria appear to produce OMVs, but the base sequences of bacteria that coexist with humans have been found to be universally free of T3SS, T4SS, and T6SS.

  However, B.I. Fragilis (or symbiotic bacteria) send beneficial microbial molecules to the immune system; The identification of mechanisms and / or molecular triggers for the fragilis (or symbiotic bacteria) to perform immunomodulatory properties continues to be challenging.

  Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, presents with a variety of symptoms, whereby uncontrollable intestinal inflammation causes serious conditions of the digestive tract. IBD afflicts about 1 million people in the United States. In addition to the medical and social burden of IBD, the incidence of illness is surprisingly increasing in 'Western' societies, and current therapies are rather inadequate. The cause of IBD remains unclear after decades of research, but defects related to intestinal bacteria in immune regulation appear to have an important role in the disease (Non-Patent Document 1).

Braun, J. & Wei, B. Body traffic: ecology, genetics, and immunity in inflammatory bowel disease. Annu Rev Pathol 2, 401-429 (2007) .8 (1) pp. 2-6

  The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, problems, and advantages may be apparent from the description, drawings, and claims.

We have shown that PSA is delivered to the host by outer membrane vesicles (OMVs), secretory structures that target bacterial molecules of the host cell. OMVs containing PSA are absorbed by dendritic cells of the host immune system. Following ingestion of OMVs, PSA regulates (programs) dendritic cells to induce differentiation of regulatory T cells (Treg) that express Foxp3 and the anti-inflammatory cytokine interleukin-10 (IL-10) . Treg cell differentiation by OMVs requires the expression of Toll-like receptor 2 (TLR2) and the production of IL-10 by dendritic cells.
In particular, purified OMVs lead to in vitro differentiation of functional Tregs with strong inhibitory activity in a PSA-dependent manner. Treating animals with OMVs containing PSA prevents experimental colitis and suppresses proinflammatory cytokine responses in the gastrointestinal tract. These discoveries reveal that symbiotic bacteria provide advantageous microbial factors through the secretion of vesicles and a process designed into a new way of delivery in probiotic therapy for IBD. To clarify.

  In one embodiment, a medicinal composition for delivering a compound to the mucosa is provided, the medicinal composition comprising an outer membrane vesicle formed by a lipid membrane surrounding an aqueous environment, and a pharmaceutically suitable carrier. And the vesicle contains a PSA ligand.

  In other embodiments, the membrane comprises an outer membrane vesicle formed of a lipid membrane surrounding an aqueous environment and comprising a PSA ligand, and a pharmaceutically suitable carrier, wherein the lipid membrane is A medicinal composition for delivering a compound to the mucosa obtained from the outer membrane of Fragilis is provided.

  In a more detailed embodiment of the composition of paragraph [0012], the PSA ligand is L-PSA.

  In yet another embodiment of the composition of paragraph [0012], the medicinal composition further comprises one or more agents or treatments known to be used in the treatment of inflammatory bowel disease. Yes.

  In one embodiment, a method of treating an inflammatory disease or inflammation in an individual administers an effective amount of a composition having an outer membrane vesicle formed by a lipid membrane surrounding an aqueous environment and comprising a PSA ligand. Have that.

  In a detailed embodiment of the method of paragraph [0016], the inflammatory disease is inflammatory bowel disease.

  In yet other embodiments, the medium for delivering the compound to the mucosa has outer membrane vesicles formed by a lipid membrane surrounding the aqueous environment and containing PSA ligands.

  In a specific embodiment of paragraph [0018], the medium further comprises a compound other than PSA.

  In another embodiment, a method for directing functional Tregs to potent inhibitory activity, utilizing an outer membrane vesicle comprising a PSA ligand formed by a lipid membrane surrounding an aqueous environment Is provided.

  In a more detailed embodiment of the method of paragraph [0020], Treg cells suppress T cell proliferation and / or further suppress immunity by reducing dendritic cell activity.

  In another embodiment of the method of paragraph [0020], the method further comprises providing DCs for co-culture, wherein IL-10 is produced and IL-10 production Is dependent on the presence of TLR2 expression in DCs, and no TLR2 expression in DCs is required compared to PSA activity alone in IL-10 DC production (no OMVs).

  In other embodiments, there is provided a method for identifying a compound that reduces inflammation or ameliorates symptoms of an inflammatory disease in vitro, the method comprising providing a candidate compound and the compound Culturing Treg cells with, further providing T cells, and determining whether said Treg cells suppress T cell activity.

  In further details of the embodiment of paragraph [0023], the method further comprises dendritic cells and / or the Treg cells express Foxp3.

  In another embodiment, a bacterial material for use as a medicament is provided, the material comprising an outer membrane vesicle formed by a lipid membrane surrounding an aqueous environment and a vesicle comprising a PSA ligand; Is included.

  In a more detailed embodiment of the drug of paragraph [0025], the bacterial substance further comprises excipients and / or one or more drugs known to be used in the treatment of inflammatory bowel disease. Contains.

  In another embodiment, a bacterial substance used in the treatment of inflammatory diseases or inflammation is provided, the material comprising an outer membrane vesicle formed by a lipid membrane surrounding an aqueous environment, and a PSA ligand. And a vesicle containing.

  In a more specific embodiment of the bacterial substance of paragraph [0027], the inflammatory disease is inflammatory bowel disease.

  In other embodiments, the composition for directing functional Treg differentiation to strong inhibitory activity has outer membrane vesicles formed by a lipid membrane surrounding an aqueous environment and comprising a PSA ligand.

  In a more specific embodiment of the composition of paragraph [0029], Treg suppresses T cell activity and / or Treg cells express Foxp3.

  Further provided are methods and systems for selecting microorganisms and related substances that have immunomodulatory properties, specifically anti-inflammatory properties. In particular, in some embodiments, methods and systems are provided that allow the identification of bacterial substances that can individually induce immune responsiveness comparable to one of Bacteroides fragilis.

  In other embodiments, methods for identifying bacterial substances having immunomodulatory properties are described. The method comprises contacting only T cells or T cells with a candidate bacterial substance in the presence of antigen presenting cells and selecting from the group consisting of IL-10, Foxp3, TGF31, TGF32, perforin, and granzyme B One or more anti-inflammatory biomarkers, and IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21 , Detecting expression of at least one of one or more inflammatory biomarkers selected from the group consisting of IL-22, IL-23, IL-17, TNFα. The method further measures the anti-inflammatory performance of a candidate bacterial substance by detecting increased expression of one or more anti-inflammatory biomarkers or decreased expression of one or more anti-inflammatory biomarkers after contact. Have to do.

  In yet another embodiment, a method for identifying a bacterial substance having immunomodulatory performance is provided. The method comprises contacting the candidate bacterial substance with antigen presenting cells and culturing the antigen presenting cells with T cells after contact. The method further comprises one or more anti-inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGF31, TGF32, perforin, and granzyme B, and IFNγ, IFNα, IFNβ, IL-1, IL -4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17, one or more selected from the group consisting of TNFα Detecting the expression of at least one of the inflammatory biomarkers. The method further measures the anti-inflammatory performance of a candidate bacterial substance by detecting increased expression of one or more anti-inflammatory biomarkers or decreased expression of one or more anti-inflammatory biomarkers after culture. Have to do.

  In another aspect, a method for identifying a bacterial substance with immunomodulatory capabilities in an animal is described. The method comprises treating a recombinant genetic marker non-human animal with a candidate bacterial substance, the recombinant genetic marker non-human animal comprising IL-10, Foxp3, TGF31, TGF32, perforin, and granzyme B. One or more labeled anti-inflammatory biomarkers selected from the group, and IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL -13, IL-21, IL-22, IL-23, IL-17, genetically to express at least one of one or more labeled inflammatory biomarkers selected from the group consisting of Has been modified. The method detects the expression of at least one anti-inflammatory biomarker of one or more in a recombinant gene marker non-human animal or the expression of at least one inflammatory biomarker of one or more after treatment. And measuring the anti-inflammatory performance of the candidate bacterial substance by detecting an increase in the expression of one or more anti-inflammatory biomarkers or a decrease in the expression of one or more inflammatory biomarkers after treatment. In addition.

  In another aspect, a system for sorting bacterial material is described. The system includes at least two T cells, an antigen presenting cell, one or more anti-inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGF31, TGF32, perforin, and granzyme B, and IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17, And a reagent for detecting at least one of one or more inflammatory biomarkers selected from the group consisting of TNFα, which are combined in the method for identifying an anti-inflammatory bacterial substance described above at the same time. Or used continuously.

  The methods and systems described above can be used in connection with medical, pharmaceutical, veterinary applications, as well as basic biological research and various applications, and can be identified by those skilled in the art in interpreting the present disclosure. In this method and system, it is desirable to investigate the immunomodulatory ability and in particular to examine the anti-inflammatory ability of the substance.

  The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate one or more embodiments of the present disclosure, and together with the detailed description and examples, illustrate the nature and practice of the disclosure. Useful.

FIG. 1 shows that outer membrane vesicles (OMVs) from Bacteroides fragilis contain PSA. FIG. 1A shows OMVs produced by wild-type B. fragilis and B. fragilis ΔPSA detected by transmission electron microscopy of B. fragilis rich in EDL (electron density layer). FIG. 1B shows an immunoblot analysis of whole cell (WC) and outer membrane vesicle (OMV) extracts from wild-type and SA-mutant bacteria. FIG. 1C shows purified OMVs immunogold-labeled, stained with anti-PSA and anti-IgG-gold colloid complex (5 nm), and analyzed with an electron microscope. FIG. 1D shows glycoprotein staining of capsular polysaccharide preparations prepared from whole cells and OMVs. FIG. 2 shows typical results showing that OMVS depends on PSA to protect animals from experimental colitis, enteritis. FIG. 2A shows a diagram reporting weight loss in a group of animals after induction of TNBS colitis (day 0), measured as a decrease from the initial weight until the day of death (day 4). All groups contain at least 4 animals and error bars indicate standard error (SEM). Results are representative of three independent trials, p values determined by one-way ANOVA are * P <0.05, *** P <0.001. FIG. 2B shows an image of the untreated intestine immediately after excision and shows length quantification (graph) from vehicle treated (ethanol) and TNBS treated groups (n = 4 / group). Error bars indicate SEM. The results are shown in 3 independent experiments performed, the p-value determined by one-way ANOVA is *** P <0.001, NS: no significant difference. FIG. 2C shows an image of the intestinal site stained with hematoxylin and eosin (H & E) representing each treatment group. FIG. 2D shows the colitis score given to animals by a blinded pathologist (GWL) according to a standard score stem (online) (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology). 1.19.1-15.19.14). Each mark represents an individual animal. Results are shown from 3 independent experiments performed independently, *** P <0.001, NS: no significant difference. FIG. 2E is a diagram showing a cytokine transcription analysis of RNA collected from the whole intestine by qRT-PCR. Error bars indicate SEM from 4 animals / group. Results are representative of 3 independent tests. FIG. 2F is a diagram showing a result of cytokine transcription analysis by qRT-PCR of RNA collected from purified CD4 + T cells of mesenteric lymph nodes. Error bars indicate SEM from 4 animals / group. Results are representative of 3 independent tests. FIG. 3 shows the results suggesting that OMVs-containing PSA induces IL-10 production and Foxp3 expression from T cells co-cultured with treated DSc. FIG. 3A shows a flow cytometry (FC) analysis of OMVs internalized by DC. OMVs were labeled with FITC (fluorescein isothiocyanate) and incubated with grown DCs for various times (as shown). Cells were stained with anti-CDl lc. The percentage indicates the number of CD11C + OMV + individuals. FIG. 3B shows FC plots of DCs cultured with WT-OMVS and ΔPSA-OMVS for various times (as shown) and stained with anti-CDllc and anti-MHCII. Percentages indicate the number of MHCII individuals in CDl lc + cells. FIG. 3C shows an ELISA analysis for IL-10 of culture supernatants from DC cells or DC-T cell co-cultures, in which DCs were targeted with OMVs for 18 hours and washed And co-cultured with or without primary CD4 + T cells. The supernatant was collected on the fourth day of culture. Media samples showed no labeling with OMVs, but were otherwise treated similarly. Anti-CD3 was added to some samples to enhance the T cell response. Error bars indicate SEM from 3 samples. Results are representative of 5 or more independent tests, * P <0.05, ** P <0.01. FIG. 3D (left) shows an ELISA analysis similar to FIG. 3C, but also includes DCs differentiated from IL-10-animals. Error bars indicate SEM from 3 samples. Results are representative of 3 independent trials, * p <0.05, NS: no significant difference. FIG. 3D (right) shows an ELISA analysis similar to FIG. 3C, but also includes DCs differentiated from TLR2 − / − animals. SEA: Staphylococcal enterotoxin A. Error bars indicate SEM from 3 samples. Results are representative of 3 independent trials, * p <0.05, NS: no significant difference. FIG. 3E shows IL-10 (left) and Foxp3 (right) transcription levels determined by qRT-PCR of RNA recovered from purified T cell subsets after in vitro culture with DCs. . Co-culture is set to (ce) on day 4, CD4 + CD25 + T cells and CD4 + CD25-T cells are purified by magnetic bead separation (purity> 95), RNA is extracted with RNeasy mini kit It was done. Relative values were normalized to [β] -actin. Error bars indicate SEM from 3 samples. Results are representative of 3 independent trials, * P <0.05, *** P <0.001, NS: no significant difference. FIG. 3F shows co-culture set to (c-e), but CD4 + T cells from Foxp3-GFP mice were used. After culturing with DC labeled OMV for 4 days, cells were stained with anti-CD4 and Foxp3 was detected by GFP expression using FC. Results are representative of two independent trials. FIG. 4 shows typical results suggesting in vitro generation of Treg inhibitory action by PSA-containing OMVS. FIG. 4A shows an FC histogram of IL-10 expression by CD4 + T cell subsets after 4 days of co-culture with OMVS treated DCs. Splenic CD4 + T cells were purified from IL-10 GFP mice, co-cultured, stained with anti-CD4 and anti-CD25, and IL-10 expression was measured by GFP expression. Percentages indicate the number of IL-10 + individuals in CD4 ++ CD25 + and CD4 + CD25- subsets. Results are representative of 3 independent tests. FIG. 4B shows the in vitro inhibition of undosed response cells by purified CD4 + CD25 + T cells after co-culture with DCs treated with medium (control), WT-OMVs, ΔPSA-OMVS. Cell proliferation was measured by CFSE diluted FC. The Treg: Teff ratio is shown and the percentage indicates the total number of proliferating cells. No Treg: Shows only CD4 + CD25 − cells. Results are representative of two independent tests. FIG. 4C shows the percentage of CD4 + T cells at each growth peak (as marked as 1, 2, 3, 4, 5 in FIG. 4B). Results are shown from 3 independent experiments performed independently, *** P <0.001, * p <0.05, NS: no significant difference. FIG. 5 shows that wild type B. fragilis and the PSA deletion mutant B. fragilis produce similar amounts of OMVs in in vitro culture. The total protein recovered from the preparation of each OMVs was normalized by the culture OD600 at the time of recovery. Error bars indicate SEM. Results were obtained from a combination of more than 10 tests performed independently. p-value is determined by Student's t test, NS: no significant difference. FIG. 6 shows that the majority of wild-type B. fragilis purified OMVs contain PSA, and immunogold labeling of PSA in purified OMVS covers a range of 10 random samples (1 μm × 1 μm) associated with PSA Of these, although not more than 60% of WT-OMVS was observed, none of the observed ΔPSA-OMVs was positively stained for PSA. (Immunogold labeling of WT-OMVs with anti-IgG-gold ensures only the specificity of the label). FIG. 7 shows that OMVs removed from wild type B. fragilis or PSA deletion mutant B. fragilis show no significant differences in protein composition. Proteomic mass spectrometry shows 100% overlap of identified proteins (more than one unique peptide that is distinguished from each protein) between WT-OMVs and ΔPSA-OMVs. Of all the identified proteins, we compared the amount of relatively abundant proteins semi-quantitatively according to the number of unique peptides each identified. Most of them show no differences made independently. Error indicates SEM. p values are determined by Student's t test, **: P <0.01, *: P <0.05, NS: no significant difference. FIG. 8 shows that actin polymerization is required for OMV uptake by DC. Flow cytometric analysis of DC internalization of OMVs was pretreated with cytochalasin D. OMVs were labeled with FITC (fluorescein isothiocyanate) and incubated with DCs grown (as indicated) for various times. Cells were stained with anti-CDl lc. The percentage indicates the number of CD11c + OMV + individuals. FIG. 9 shows that WT-OMVs or ΔPSA-OMVs are internalized and localized in the cytoplasm of DCs. The fluorescence micrograph shows the internalization of OMV (WT or ΔPSA) by DC. OMVs were labeled with FITC (grey arrows) and incubated with grown DCs for 2 hours. The cells were fixed and the cell membrane was stained with wheat germ agglutinin (WGA) -tetramethylrhodamine (black arrow). Scale bar: 7.5 μm. FIG. 10 shows that WT-OMVs and ΔPSA-OMVs upregulate costimulatory molecules for DC activation. FC plots of DCs cultured with WT-OMVs and ΔPSA-OMVs for various times (as indicated) and stained with anti-CD11c and anti-CD86. Percentages indicate the number of CD86 + individuals in CD11c + cells. FIG. 11 shows PSA-induced expression of biomarkers in the embodiments described herein. The data shown in each figure is representative of three independent experiments. White bars represent cells from PBS-treated mice and gray bars represent cells from PSA-treated mice.

  Described herein are suitable media for delivering compounds to the mucosa.

  As used herein, the terms “wild type OMV”, “OMVs-containing PSA”, and “PSA-OMV” are used interchangeably.

  The term “medium” within the meaning of the present disclosure indicates a medium in which a compound is given and in which it is expressed, manifests and / or exhibits properties. Typical media include some of the various media that normally act as solvents, carriers, or binders for the active ingredients, particularly those associated with the drug.

  The terms “send” and “ship” associated with a compound or substance within the meaning of this disclosure indicate that the compound or substance moves from its original location to the target location, specifically one or more. To the position formed by each of the cells, tissues, or organs. Specifically, for purposes of this specification, a compound is defined as a compound that is moved so that at least one activity associated with the delivered compound functions in each of one or more cells, tissues, or organs. Sent.

  As used herein, the term “compound” refers to various chemical substances composed of one or more chemical elements, and is not limited to biomolecules, particularly various substances, molecules, including drugs, or Contains ingredients. The term “biomolecule” as used herein refers to saccharides, amino acids, peptide proteins, oligonucleotides, polynucleotides, polypeptides, organic molecules, haptens, epitopes, biological cells, biological cells. A substance compound or component related to biological activity including, but not limited to, vitamins and hormones. As used herein, the term “drug” refers to a substance that, when adsorbed to an organism, alters normal body function. For the purposes of this disclosure, a drug is specifically a chemical used in the treatment, treatment, prevention, or diagnosis of a disease, or separately used to increase physical or mental health. Includes chemicals to be made.

  For example, if the PSA is a 'compound' as described above, different compounds can be further delivered by the vesicles in addition to the PSA.

  The term “mucosa” within the meaning of the present disclosure refers to the intima that is almost endodermal and extends to the surface in the epithelium and is involved in absorption and secretion. Each mucosa is located at the boundary with various body cavities exposed to the external environment and internal organs. The mucous membrane is connected to the skin in various places such as the nose, mouth, lips, eyelids, ears, genitals, and anus.

  In the compositions described herein, and related methods and systems, the medium is formed by vesicles containing one or more compounds to be delivered.

  A “vesicle” within the meaning of this disclosure is a supramolecular complex formed by membrane-forming lipids and further molecules assembled in an aqueous environment. Specifically, in the vesicles described herein, membrane-forming lipids are placed in a lipid layer that surrounds an internal aqueous environment, also shown as cytoplasm.

  As used herein, the term “film-forming lipid” or “amphipathic lipid” refers to a collection of both hydrophilic and hydrophobic lipids that form a lipid layer structure in an aqueous environment, The lipid layer structure consists of either one layer of amphiphilic molecules known as polar lipids or two layers in which the amphiphilic molecules face in opposite directions. Each polar molecule has a hydrophilic portion, that is, a polar group such as a derivatized phosphate group or sugar group, and a hydrophobic portion, that is, a long-chain hydrocarbon. Illustrative polar lipids include phospholipids, sphingolipids, glycolipids, ether lipids, sterols, and phosphocholines. Amphipathic lipids include but are not limited to membrane lipids, ie phospholipids such as dimyristoyl phosphatidylcholine (DMPC), dioleoylphosphoethanolamine (DOPE), or dioleoylphosphatidylcholine (DOPC), or biological Examples include amphipathic lipids that constitute the membrane. In an embodiment, the small membrane is formed by a lipid bilayer similar to a cell membrane (a biofilm that separates the cell interior from the external environment and surrounds the aqueous environment), a specific example being the outer membrane of B. fragilis. .

  The vesicles described herein also include lipopolysaccharide (LPS) associated with the membrane of the vesicle or contained in the aqueous environment of the vesicle. As used herein, the term “lipopolysaccharide” refers to a macromolecule composed of covalently linked lipids and polysaccharides, which are located in the outer membrane of gram-negative bacteria and are endotoxins (endotoxins). ) And cause a strong immune response in animals. As a specific example, the vesicles described herein comprise one or more LPS of B. Fragilis identified by those skilled in the art.

  The vesicles described herein may also include peptidoglycans associated with the membrane of the vesicle or contained in the aqueous environment of the vesicle. As used herein, the term “peptidoglycan” refers to a polymer compound composed of saccharides and amino acids, and a mesh-like layer is formed on the outer side of the cell membrane of bacteria (eubacteria, not archaea). It forms and forms a cell wall. As a specific example, the vesicles described herein comprise one or more peptidoglycans of B. Fragilis identified by those skilled in the art.

  Additional compounds that can be included in the vesicles include membrane proteins, membrane glycolipids, nucleic acids, in particular, B. Fragilis membrane proteins, membrane glycolipids, nucleic acids.

  Typical vesicles within the meaning of the present disclosure include a small sac covered with a membrane capable of storing or transporting material. Vesicles can be formed spontaneously due to the properties of membrane-forming lipids, or can be prepared from bacterial membranes. Most vesicles have specific actions depending on the substances contained in the membrane and / or the substances contained in the aqueous environment.

  In certain embodiments, the vesicles described herein are formed by a portion of a bacterial membrane. In certain embodiments, the vesicles are formed by bacterial outer membrane vesicles (OMVs).

  In certain embodiments, the vesicles described herein are formed by a portion of a B. Fragilis membrane, in particular formed from a B. Fragilis OMV, as illustrated in the Examples. Has been.

  In some embodiments, the delivered compound comprises a drug, a candidate drug, a dietary supplement (one or more ingredients (vitamins or amino acids) that are intended to assist in dietary restrictions or are not considered food) Oral products) or other compounds that are desired to be transported to the mucosa. These one or more compounds may be included in a suitable amount in the vesicles described herein. An appropriate amount can be determined by one skilled in the art depending on the particular compound being delivered.

  In one embodiment, it comprises one or more zwitterionic polypolysaccharides, in particular polypolysaccharide A.

  As used herein, the term “amphoteric polypolysaccharide” refers to a synthetic or natural polymer compound having one or more monosaccharides linked by a glycosidic bond, Having at least one positively charged portion and at least one negatively charged portion. The amphoteric polypolysaccharide is not limited, and includes macromolecules of all lengths, from monosaccharide or disaccharide polymer compounds to polymer compounds containing hundreds to thousands of monosaccharides. In some embodiments, the zwitterionic polypolysaccharide comprises repeating units, each repeating unit having 2 to 10 monosaccharides, a positively charged moiety (eg, a free positively charged amino moiety), and a negatively charged Moieties (such as sulfonate, sulfate, phosphate, and phosphonate moieties). In some embodiments, ZPs have a molecular weight between 500 Da and 2,000,000 Da. In some embodiments, ZPs have a molecular weight between 200 Da and 2500 Da. Typical ZPS include, but are not limited to, PSA and PSB from Bacteroides Fragilis, CP5 / CD8 from Staphylococcus aureus, and Sp1 / CP1 from Streptococcus pneumonia. Zwitterionic polysaccharides can be isolated from natural resources, in particular from bacterial resources, in particular by purification. Zwitterionic polysaccharides can be identified and prepared by those skilled in the art by chemical or biochemical methods and by microbial genetic engineering techniques. Accordingly, these methods and techniques are not described in further detail herein.

  As used herein, the expression “polypolysaccharide A” (or PSA, PSA ligand) refers to molecules produced by the PSA locus of Bacteroides fragilis and its derivatives, which are limited. The polymer of repeating units {→ 3) α-d-AAT Galp (l → 4)-[β-d-Galf (1 → 3)] α-d-GalpNAc (l → 3)-[4,6- Pyruvate] β-d-Galp (l →}, where AATGal is acetamido-amino-2,4,6-trideoxygalactose, galactopyranosyl residues are 0-4 and 0-6 The term “derivative” as used herein for a first polypolysaccharide (eg, PSA) is structurally related to the first polypolysaccharide. A second polysaccharide that is related, the second polysaccharide having characteristics that are not present in the first polysaccharide. The polysaccharide derivative of PSA is derived from the first polypolysaccharide by modification that maintains the functional properties of the first polypolysaccharide that is awakened. However, the modification may be related to additional functions that are not present in the original polypolysaccharide, or may not be related, however, the modification of the PSA polypolysaccharide. The derivatives maintain one or more of the active activities associated with the PSA described herein that are associated with the anti-inflammatory activity of PSA.

  In some embodiments, the low molecular weight PSA (L-PSA) is between 70 kDa and 200 kDa, and the high molecular weight PSA (H-PSA) is greater than 200 kDa.

  In certain embodiments, the mucosa is a mucosa of an individual gastrointestinal tract, in particular the intestinal mucosa, gastric mucosa, esophageal mucosa, buccal mucosa, oral mucosa and / or buccal mucosa.

  In certain embodiments, the vesicles described herein may be used in connection with means and applications such as those included as Appendix A and described in US Provisional Application S / N 61 / 321,527. This description forms part of the present disclosure and is incorporated herein by reference in its entirety.

  In certain embodiments, the vesicles described herein may be used in connection with methods and systems such as those included as Appendix B and described in US application S / N 12/766787. This description forms part of the present disclosure and is incorporated herein by reference in its entirety.

  In certain embodiments, the vesicles described herein are used in a method of delivering the compound to individual mucosa. As a specific example, the method comprises contacting a vesicle containing the compound with a mucous membrane for a while in an environment where a substance can contact the membrane. In the method, the vesicle is formed by a lipid membrane surrounding the aqueous environment and the compound to be delivered is contained in the aqueous environment of the vesicle.

  In another embodiment, the composition or medicinal composition comprising the vesicle or OMV is contacted with the mucosa for a while in an environment where the substance can contact the membrane.

  As used herein, the term “contact” or “culture” refers to an environment in which at least one of an interactive or non-interactive action or effect between two things is exerted. Shows an act that causes a spatial relationship between two things to be provided for some time. In particular, the culturing takes place between the vesicle and the mucosa and may cause direct contact and / or interaction between the vesicle and the mucosa, or indirectly of the vesicle. After action (eg, after the action or modification of other substances that react directly with the membrane), mucosal modification will occur.

  Appropriate conditions for contacting or culturing are determined by one skilled in the art in accordance with the description of the present disclosure in terms of the compound, the mucosa, and the experimental design.

  In certain embodiments, the vesicles herein can be included in a composition for delivering a compound to the mucosa. The composition comprises one or more vesicles as described herein, optionally combined with a further medium that acts as a vesicle solvent, carrier, binder, or diluent.

  The term “condition” —as used herein—when referring to an animal or human situation—usually refers to the physical condition of an individual body, generally in whole or in one or more parts, The physical status does not match the physical status of the individual body in whole or in one or more parts associated with a complete physical, mental and possibly socially sound situation. The conditions described herein include, but are not limited to, disorders and diseases, and the term “impair” refers to biological conditions related to functional abnormalities in the body or any part of the body. The term “disease” refers to a condition of the body that impairs the normal functioning of the body or any part of the body and is typically manifested by characteristic signs and symptoms. Typical conditions include, but are not limited to, injuries, disorders, diseases (including mental and physical disorders), syndromes, infections, unusual behavior, function and structure in individual bodies or parts thereof There are atypical changes.

  In particular, in embodiments where the compound is used as an anti-inflammatory compound, the condition can be any condition associated with inflammation or an inflammatory response. Or it can be a condition described as an inflammatory disease itself. As used herein with respect to two things, the expression “related” refers to a relationship between two things such that the occurrence of the first is concomitant with the occurrence of the second, including a limitation Not including causal and sign / symptom-disease relationships.

  Conditions associated with inflammation (or inflammatory disease) in humans include, but are not limited to, inflammatory bowel disease, including but not limited to Crohn's disease, ulcerative colitis, asthma, dermatitis, rheumatoid arthritis, severe Examples include myasthenia, Graves' disease, multiple sclerosis (MS), and psoriasis. One skilled in the art can identify those having the above-mentioned diseases using appropriate diagnostic criteria.

  In some embodiments, compositions comprising OMVs containing PSA, medicinal compositions can be used for the prevention of inflammatory diseases. Such diseases include, but are not limited to, inflammatory bowel diseases (including Crohn's disease, ulcerative colitis), asthma, dermatitis, rheumatoid arthritis, myasthenia gravis, Graves' disease, multiple sclerosis, psoriasis .

  As used herein, the term “treatment” or “treated” refers to any work, whether medical treatment or not, or medical treatment in animals or humans. Or it involves surgical conditions. Such treatment can be performed by medical or non-medical workers.

  In certain embodiments, the compositions may be administered individually and the composition may be a medicinal composition. The composition also includes one or more vesicles each containing PSA. In a more specific embodiment, the medicinal composition comprises one or more vesicles each containing PSA, one or more other compounds, and / or a pharmacologically possible or suitable carrier / vehicle. Including.

  In other embodiments, the above-described pharmaceutical composition comprising one or more vesicles each containing PSA, one or more other compounds, and / or a pharmacologically possible or suitable carrier / vehicle, In an individual / subject with an inflammatory condition or inflammation given the composition, an improvement is shown.

  In certain embodiments, the vesicles described herein can be included in a pharmaceutical composition with excipients or diluents. In particular, in certain embodiments, the pharmaceutical composition is used herein in combination with one or more compatible and pharmaceutically suitable vehicles, particularly pharmaceutically suitable diluents or excipients. Including the described vesicles.

  As used herein, the term “excipient” refers to an inert substance used as a pharmaceutically possible or suitable carrier for the active ingredient of the drug. Suitable excipients for the pharmaceutical compositions disclosed herein include any substance that enhances the ability of an individual body to absorb the vesicles herein. Suitable excipients include those that can be used to increase the volume of the formulation so that the formulation with vesicles described herein is easy to use and has an accurate dosage. In addition to the use of excipients in a single dose, excipients can also be used in the manufacturing process to facilitate handling of the vesicles described herein. Various excipients may be used depending on the route of administration and drug preparation. Typical excipients include, but are not limited to, anti-adhesives, binders, coating disintegrants, fillers, flavorings (such as sweeteners) and colorants, glidants, lubricants, preservatives, An adsorbent is mentioned.

  Pharmaceutically acceptable or suitable carriers can be, but are not limited to, organic or inorganic, solid or liquid excipients. The excipient is suitable for selected modes of application such as oral application and injection, and is administered in the form of a general pharmaceutical preparation. Examples of such preparations include solid substances such as tablets, granules, powders and capsules, and liquid substances such as solutions, emulsions and suspensions. Examples of the carrier include starch, lactose, glucose, sucrose, dextrin, cellulose, paraffin, fatty acid glyceride, water, alcohol, gum arabic and the like. If necessary, adjuvants, stabilizers, emulsifiers, lubricants, binders, pH adjusters, isotonic agents, and other common additives can be added.

  Pharmaceutically acceptable or suitable carriers are other compounds known to be suitable for intestinal abnormal situations or suitable for edible compositions (eg, antioxidants such as vitamin C, vitamin E, Selenium or zinc). Edible compositions include, but are not limited to, milk, yogurt, tofu, cheese, fermented milk, milk-based fermented products, ice cream, fermented cereal-based products, milk-based powder, infant formula, tablets, liquid Examples include bacterial suspensions, dry oral supplements, or wet oral supplements.

  As used herein, the term “diluent” refers to a substance for dilution utilized to dilute or carry the active ingredient of the composition. Suitable diluents include any substance that can reduce the viscosity of a pharmaceutical formulation.

  In certain embodiments, the compositions, compounds, and particularly medicinal compositions are prepared for intestinal administration, including, but not limited to, i) oral as tablets, capsules, or drops (Via the mouth), ii) by intestinal feeding tube, duodenal feeding tube, or gastrostomy, and iii) by rectal suppository.

  In certain embodiments, vesicles comprising a PSA as described herein can be used in a method of treating or preventing a situation in each individual.

  The method includes providing each individual with an effective amount of the composition or medicinal composition. As used herein, the term “each individual” refers to a single biological organ, including, but not limited to, inflammation in animals, particularly higher animals, particularly vertebrates such as mammals, and particularly humans. Occurs.

  “Effective amount”, “effective amount”, or “effective amount for X” means that when a compound is administered, it partially cures, eliminates, or reduces one or more symptoms of the disease in need of treatment. Or the amount of a compound, composition or medicinal composition that is effective to ameliorate or is effective to delay the onset of clinical indicators or symptoms of a disease in need of prevention. Thus, for example, an effective amount is an amount that exhibits the 'improving' action of the compound / composition / medicinal active ingredient, as shown below.

  An “effective amount” can be determined experimentally by experimenting with compounds related to in vitro and in vivo model systems known for the disease in need of treatment. Effective amounts will vary with each individual's weight, sex, age, and medical history, and will also vary with patient severity, type of disease, mode of administration, and the like. Effective amounts are readily determined by performing routine experiments, eg, titration (administering in increasing doses until an effective dose is found) and / or refer to an amount effective for a past patient. Determined by. In general, an effective amount of the present invention is administered at a dose ranging between about 0.1 mg / kg and about 20 mg / kg relative to the patient's body weight.

  As used herein, a “prophylactically effective amount” is sufficient (or other therapy) to prevent or reduce the worsening, recurrence, or onset of one or more symptoms associated with a disease. The amount of the compound / composition / medicinal composition (sufficient to improve or improve the prophylactic action of the other prophylactic agent, for example).

  It is further expected that one or more known drugs known to be useful in the treatment or prevention of inflammatory diseases, wherein the compounds / compositions / medicinal compositions of the present invention are separate or combined. It can be used in conjunction with.

  For example, the compounds / compositions / medicinal compositions of the present invention include, but are not limited to, sulfasalazine (Asalphidin), mesalazine (Asacol, Pentasa), immunosuppressants (Imlan, 6-MP, cyclosporine), methotrexate, TNF-α inhibition It can be combined with one or a combination of drugs / treatments known to be useful in the treatment of IBD such as agents (Remicade, Humira) and corticosteroids (Encorto, prednisone). Other treatments (experimental) for ulcerative colitis include aloe vera, butyric acid, boswellia, probiotics, antibiotics, immunosuppressive therapy, and nicotine.

  For example, the compounds / compositions / medicinal compositions of the present invention include, but are not limited to, Avonex (product name), Betaselon (product name), and Copacson (product name), Lebif (product name), Extabia (product name), One of the drugs / treatments known to be useful in the treatment of MS, such as Novantrone (product name) (mitoxantrone), Taisaburi (product name) (natalizumab), and Gilenya (product name) (fingolimod) Can be combined with a combination. Other drugs include intravenous immunoglobulin (IVIG) therapy, methotrexate, azathioprine (Imlan (product name)), and cyclophosphamide (cytoxan (product name)); corticosteroids; Phosphamide), Imran (product name) (azathioprine), methotrexate, plasma exchange, pulse solution medrol (product name) (IV methylprednisolone); prednisone, decadron (product name) (dexamethasone); medrol (product name) (oral) Methylprednisolone); plasma (plasma exchange), intravenous immunoglobulin (IVIG) therapy.

  In other embodiments, these other drugs can be delivered or exerted within the OMVs themselves.

  “Improved”, “improved”, and other grammatical variations herein include any beneficial changes in the individual / object that result from the treatment. An advantageous change is that the patient's condition is better than when there is no treatment. “Improved” includes, but is not limited to, preventing undesirable situations, slowing the rate of worsening of the situation, delaying the progression of undesirable situations, restoring the original normal situation, Causing the patient to maintain greater recovery or longer recovery than if not (i.e., the patient has less (or no) inflammation, or the human is usually in a symptom-free state), This includes reducing the number and / or extent of relapses and reducing the progression of new sites observed in magnetic resonance imaging (MRI) images.

  More specifically, improvements in IBD include, but are not limited to, abdominal cramping and pain, bloody stool, diarrhea, emergency with bowel movement, fever, loss of appetite, weight loss, mucous feces, colon ulceration, and anemia Reducing the severity or persistence in one or more clinical IBD symptoms such as (due to blood loss). For example, improvements in MS include, but are not limited to, visual impairment, numbness, dizziness / rotational dizziness, bladder and bowel dysfunction, weakness, tremors, movement disorders, sexual dysfunction, non-stabilization, spasticity (stiffness of legs) ), Reducing the severity or persistence in one or more clinical MS symptoms such as dysphagia, chronic tingling pain, depression, mild cognition, memory difficulties.

  Furthermore, "improved", "improved", and other grammatical variations include any change that reduces the severity, persistence, and / or the risk of developing inflammatory disease complications. In IBD, for example, 'improvement' means reducing the risk in the IBD object, and progressing in the IBD object includes a large amount of bleeding from the ulcer, intestinal perforation (rupture), stenosis, There are obstructions, fistulas (abnormal passages) and perianal disease, toxic megacolon (acute non-occlusive dilatation of the large intestine), and malignant tumors (eg colon cancer).

  In certain embodiments, at least two of the membrane-forming lipids, vesicles, PSA and one or more additional compounds to be delivered are optionally combined with other suitable drugs used in the methods described herein. Is captured in the system.

  The system can be provided in the form of a kit of parts. In a kit of parts, membrane-forming lipids, vesicles, PSA, adduct compounds, drugs, and other drugs can be included in one or more compositions. Alternatively, each of the membrane-forming lipids, vesicles, PSA, adducts, drugs, and other drugs can be included in the composition along with a suitable vehicle.

  Additional components include labeled molecules, particularly labeled capture agents that are specific for anti-inflammatory or inflammatory biolabels, or for molecules associated with their expression, or Examples include microfluidic chips, standards, and additional components identified by those skilled in the art with reference to the disclosure.

  As used herein, the term “capture agent” refers to a compound that specifically binds to a target. As used herein, the expression “specifically”, “specific”, or “specific” in relation to binding of a first compound to a second compound refers to the first compound and Each stable complex of the second compound, with other molecules present, substantially without recognizing, contacting, and structuring, the first compound and the second compound It means recognizing a stable complex of compounds, contacting, and forming a structure. Specific exemplary bindings are antigen-antibody interactions, cell receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions, and the like. What is indicated by a “stable complex” is a complex that is usually detectable, but does not require any level of stability, although greater stability is preferred. In certain embodiments, the kit comprises a labeled polynucleotide or a labeled antibody.

  Kits are provided with the appropriate instructions and other necessary reagents to perform the methods described herein. The kit typically has the composition in a separate container. For example, an instruction manual recorded and recorded on an electronic medium such as a tape or a CD-ROM or paper to perform an experiment is usually provided in a kit. The kit may also include packaged reagents and materials (ie, wash buffer, etc.) depending on the particular method used.

  As used herein, the term “immunomodulation” refers to the ability to promote conditions associated with the absence of an inflammatory response. Certain immunomodulatory performances have anti-inflammatory properties, and the term anti-inflammatory relates to properties of substances or treatments that suppress or reduce inflammation.

  As used herein, the term “inflammation”, “inflammatory condition”, or “inflammatory response” refers to a complex biological response of an individual vascular tissue to a harmful stimulus, such as a pathogen, damaged cell, or irritant. Including the secretion of cytokines, more specifically the secretion of pro-inflammatory cytokines, ie the secretion of cytokines produced mainly by activated immune cells such as microglia and involved in the amplification of the inflammatory response. Exemplary pro-inflammatory cytokines include, but are not limited to, IL-1, IL-6, TNFα, IL-17, IL21, and IL23. Typical inflammation includes acute inflammation and chronic inflammation. The expression “acute inflammation” as used herein is a short term characterized by typical signs of inflammation (swelling, redness, pain, fever, loss of function) due to infiltration in tissues by plasma and leukocytes. Show the process. Acute inflammation is typically triggered while the stimulus is present, until the stimulus is removed and settled and protected by scarring (fibrosis). As used herein, the expression “chronic inflammation” refers to a situation characterized by co-active inflammation, tissue destruction, and repair activity. Chronic inflammation is not characterized by the typical signs of acute inflammation described above. On the other hand, chronically inflamed tissues are characterized by the invasion of mononuclear immune cells (monocytes, macrophages, lymphocytes, plasma cells), tissue destruction, and repair activities including angiogenesis and fibrosis. It is done.

  As used herein, the term “substance” refers to a specific or well-defined chemical structure. The term “bacterial material” refers to those derived from bacteria, such as live bacteria, dead bacteria, and in particular, heated dead bacteria, bacterial extracts, purified molecules of bacterial origin, combinations of purified molecules of bacterial origin, There are vesicles containing one or a combination of purified molecules of bacterial origin, or purified vesicles of bacterial origin. Specifically, the bacterial material is formed by or includes outer membrane vesicles (OMVs), the outer membrane vesicles being vesicles removed from the bacterial surface and comprising a set of cargo molecules To the remote target cell. More specifically, the bacterial material is formed by or comprises outer membrane vesicles (OMVs) of B. Fragilis, and more specifically, such OMVs are further comprised of PSA. ing.

  Bacterial materials include materials from the same bacteria and materials from two or more different bacteria.

  The term “bacteria” as used herein refers to a large population of microorganisms that are unicellular and prokaryotic, typically several μm in length, and have various shapes. Specifically, bacteria within the meaning of the present disclosure include bacteria of the human flora, ie, living in human tissues and body fluids such as the deep layers of the skin in the saliva, mouth or vaginal mucosa, digestive tract, and the surface. A population of microorganisms is included. Bacterial flora includes intestinal flora (bacteria detected in human digestive tract), vaginal flora (bacteria detected in fibromuscular tubular pathway leading from uterus to outside of human body), skin bacteria A flora (bacteria detected in human skin). More specifically, the bacterium in the meaning of the present invention is an anaerobic Bacillus bacterium which is formed by one or more species of bacteroidetes of the intestinal flora, in particular a gram-negative genus and which does not form spores. Formed by one or more of the Bacteroides symbiotic to humans identified by those skilled in the art. A typical Bacteroides is B. fragilis.

  As used herein, the term “recombinant genetic marker non-human animal” refers to an animal containing any of a variety of genetic engineering techniques or a naturally transformed non-natural genetic material. Such non-natural genetic material (gene transfer) acts as a “biomarker” (as defined herein) and / or has the ability to produce RNA or protein in non-human animals. I have it.

  As used herein, the term “T cell” refers to a subpopulation of lymphocytes (white blood cells or leukocytes) that have been identified by one skilled in the art and include different cell types. T helper cells in the present disclosure include effector Th cells (such as Th1, Th2, and Th17), ie, Th cells that secrete cytokines, proteins, or peptides that stimulate or react with other leukocytes. Also, suppressor Th cells (such as Treg), ie, Th cells that suppress the immune mechanism and thereby maintain immune system homeostasis and resistance to self-antigens.

  As used herein, the term “antigen-presenting cell” refers to a cell that exhibits a foreign antigen complex along with a major histocompatibility complex (MHC) on its surface. Specifically, antigen presenting cells include dendritic cells, macrophages, B cells, and other cells identified by those skilled in the art.

  As used herein, the term “contact” or “culture” is used for a while in an environment in which at least one of an interactive or non-interactive action or effect between two things is exerted. Figure 2 illustrates an act that causes the occurrence of a spatial relationship between two things provided during. In particular, the culturing is performed between the cellular material and the cell, which may cause direct contact and / or interaction between the cellular material and the cell, or indirect cellular material. After action (eg, after the action or modification of another substance that reacts directly with the cell), it will cause modification of the cell.

  “Treatment” or “treatment” can be effected by local or systemic administration of a bacterial substance, compound, composition, or pharmaceutical composition. Systemic administration includes intra-gastrointestinal administration (eg, oral administration, gastric feeding tube, duodenal feeding tube, gastrostomy, enteral feeding, rectal administration) and parenteral administration (eg, intravenous administration, intraarterial administration). , Intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical injection). Typical administrations include, but are not limited to, transdermal administration, inhalation administration (such as an asthma drug), enema, eye drops (on the conjunctiva), ear drops, intranasal route (eg, decongestant nasal drops) And vaginal administration.

  Similarly, individuals with such inflammatory diseases, including but not limited to Crohn's disease, ulcerative colitis, asthma, dermatitis, rheumatoid arthritis, myasthenia gravis, Graves' disease, multiple sclerosis, psoriasis The amount of medicinal composition provided is determined empirically by those skilled in the art.

  A typical culture of antigen presenting cells with T cells comprises antigen presenting cells by adding purified antigen presenting cells to cultured cells of T cells or by adding purified T cells to cultured cells of antigen presenting cells. It is performed in vitro by mixing cultured cells and cultured cells containing T cells. Further exemplary cultures in vivo with T cells and antigen presenting cells include transplanting antigen presenting cells into a tissue of an individual containing T cells or transplanting T cells into an individual tissue containing antigen presenting cells. Have. In certain embodiments, the individual is a non-human transgenic animal that has been genetically modified to express a labeled inflammation or anti-inflammatory biomarker.

  Further manipulations and techniques of contacting the substance with T cells or antigen presenting cells and culturing antigen presenting cells and T cells in vitro or in vivo are identified by one of ordinary skill in the art with reference to this disclosure. .

  In the methods and systems described herein, detection of expression of inflammatory or anti-inflammatory biomarkers is performed in vitro or in vivo by techniques identified by those skilled in the art, such as labeled biolabels, etc. It has the use of a label molecule or a label molecule specific for a biolabel or related molecules.

  As used herein, the term “detecting” or “detection” is limited to the presence of an analyte or associated signal, or the fact of the presence, to a test sample, reaction mixture, molecular complex, substance. It shows that it measures in a part of the space that includes them. Detection refers to, but is not limited to, a “quantitative” measurement of the amount or mass (also referred to as quantification) of an analyte or associated signal, which is related to or includes the measurement. Any analysis designed to measure an analyte or related signal quantity or concentration is included. The detection is “quantitative” indicating, or related to, or including the identification of the amount or type of the analyte or related signal relative to the abundance of other analytes or related signals that are not quantified. Is.

  The terms “label” and “labeled” as used herein as a component or molecule of a complex relate to a detectable molecule, including but not limited to radioisotopes, fluorescent dyes Molecules, chemiluminescent dyes, color formers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (eg, biotin, avidin, streptavidin or haptens) It is done. The term “fluorescent dye molecule (fluorophore)” relates to a substance or part thereof that can fluoresce in a detected image. Thus, as used herein, the term “signal” or “label signal” refers to a signal emitted from a label that allows detection of the label, including but not limited to radioactivity, Examples include fluorescence, chemiluminescence, and the formation of compounds as a result of enzyme reactions.

  Detection can be performed by detecting the degree of expression of the biolabel, its precursors or analogs, and / or analytes associated therewith. As used herein with respect to two things, the expression “related” indicates a relationship between two things such that the occurrence of the first one is related to the occurrence of the second, Non-limiting examples include causal relationships and signs / symptoms-disease relationships. Specifically, the detection is performed qualitatively or quantitatively, detecting molecules such as RNA, proteins, precursors thereof, different types (ie, mRNA, tRNA, and rRNA), degradation products, and / or , Including the detection or measurement of properties associated therewith. Techniques and operations for performing detection are identified by one of ordinary skill in the art with reference to the description of the disclosure.

  Exemplary methods for detecting biomarker expression include, but are not limited to, methods known to those skilled in the art such as ELISA, Q-PCR, and detection of intracellular cytokine staining by FACS. In some embodiments, the expression of the biomarker is detected by fluorescence-based measurement information in cultured cells performed using an antibody specific for the biomarker or related molecule, wherein the antibody is a fluorochrome molecule. Labeled and such antibodies include, but are not limited to, low molecular dyes (dyes), protein chromophores, particle dots, and gold microparticles. In certain embodiments, the expression of the biolabel is detected by detecting the expression of the label in vivo (eg, animal tissue) or in vitro (cultured cells) in the transcriptional control of the biolabel promoter. In some of these embodiments, the biolabel can specifically be IL-10 or Foxp3. Further techniques will be identified by one of ordinary skill in the art upon contact with the description of the present disclosure and will not be described in further detail.

  In the methods and systems described herein, the anti-inflammatory ability of a candidate bacterial substance is increased expression of one or more anti-inflammatory biomarkers, or one or more inflammations after contact and / or culture. It can be measured by a decrease in the expression of the biomarker. The measurement of the increase and / or decrease of the biomarker is based on the detected expression of the inflammatory biomarker after contact and / or culture and the pre-detected expression in the expression of the same biomarker in the absence of contact and / or culture. This is done by comparison. Measurement of the increase and / or decrease in biomarker expression is performed by comparing the expression of the detected label with the detected expression of the same biomarker in the control cells in the absence of contact and / or culture.

  Specific examples are provided in the Examples, where the expression of anti-inflammatory biomarkers IL-10, Foxp3, TGF31, TGF-32, perforin, and granzyme B is increased and the inflammatory biomarkers are A decrease in the expression of TNFα or IL-17A has been associated with the mechanism of action of Bacteroides fragilis, which is related to the relationship between the above-mentioned expression type and the biological activity of Bacteroides fragilis, in particular the anti-inflammatory action. Supports the relationship. The anti-inflammatory activity of Bacteroides fragilis has been identified by those skilled in the art and has been described in various publications and patent publications, including but not limited to US05679654, US07083777, US2004092433, WO07092451, and WO2009062132. Which are incorporated herein by reference in their entirety. The specific anti-inflammatory activity of the candidate bacterial substance is based on the measurement of the specific set of cytokines produced and also on the further measurement of cells activated by the candidate bacterial substance identified by the methods described herein Can be measured by those skilled in the art.

  In certain embodiments, detecting an increase in the expression of anti-inflammatory or detecting a decrease in the expression of an inflammatory biomarker is an anti-antigen such as IL-10, Foxp3, TGF31, TGF32, perforin, and granzyme B, or combinations thereof. The ability of the substance to induce inflammatory biomarkers, or IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, The performance of substances that suppress inflammatory cytokines, such as IL-22, IL-23, IL-17, TNFα or combinations thereof, is shown.

  In certain embodiments, the method comprises contacting candidate bacterial material in vitro or in vivo with T cells in the presence of T cells alone or antigen presenting cells, and IL-10, Foxp3, TGF31, TGF-32 , Detecting the expression of at least one anti-inflammatory biomarker selected from the group consisting of perforin, and granzyme B, wherein the method comprises increasing the expression of the anti-inflammatory biomarker after contact. Shows the anti-inflammatory performance of the candidate bacterial substance. Specifically, in some of these embodiments, expression of IL-10, Foxp3, TGF31, TGF-32, perforin, and / or granzyme B is expressed in Foxp3-expressing T cells, more specifically in Foxp3 expression. Performed in Treg cells.

  In certain embodiments, the method comprises contacting the candidate bacterial substance with the antigen-presenting cell and culturing the antigen-presenting cell with the T cell after contact. The method further comprises detecting the expression of at least one anti-inflammatory biomarker selected from the group consisting of Foxp3, IL-10, TGF31, TGF32, TGF32, perforin, and granzyme B; In, detection of increased biomarker expression after culture indicates the anti-inflammatory performance of the candidate substance. Specifically, in some of these embodiments, expression of IL-10, perforin, and / or granzyme B is performed in Foxp3-expressing T cells, more specifically, Foxp3-expressing Treg cells.

  In certain embodiments, the method comprises contacting a candidate bacterial substance with a T cell alone or in the presence of an antigen presenting cell, and an anti-inflammatory organism selected from the group consisting of IL-17 and TNFα. Detecting the expression of at least one label, wherein a decrease in expression of the inflammatory biomarker after contact indicates the anti-inflammatory performance of the candidate bacterial substance.

  In certain embodiments, the method comprises contacting the candidate bacterial substance with antigen presenting cells and culturing the antigen presenting cells with T cells after contact. The method further comprises detecting the expression of at least one inflammatory biomarker selected from the group consisting of IL-17 and TNFα, wherein the method reduces the expression of the inflammatory biomarker after contact. Shows the anti-inflammatory performance of the candidate bacterial substance.

  In certain embodiments, the candidate bacterial material comprises bacteria in a pure system. In other embodiments, the candidate bacterial material comprises bacteria in a mixed system. The bacterium can be a live bacterium, a dead bacterium, a simple extract or product from the bacterium, or a combination thereof. The bacteria can be from a laboratory, from a repository such as ATCC, isolated from an animal, isolated from a human, or a combination thereof.

  In some embodiments, the animal isolate is isolated from dung. The isolate can be intestinal contents or a combination of dung and intestinal contents. In certain embodiments, the isolate from human is isolated from stool. The isolate can be intestinal contents or a combination of stool and intestinal contents.

  In certain embodiments, the method comprises treating a recombinant genetic marker non-human animal, specifically a recombinant genetic marker mammal such as a mouse or rat, with a candidate substance, and a recombinant genetic marker animal after treatment. Detecting the expression of a biomarker in The recombinant genetic marker non-human animal expresses a labeled anti-inflammatory or inflammatory biomarker selected from the group consisting of IL-10, Foxp3, TGF31, TGF-32, perforin, granzyme B, and IL17, TNFα. Genetically modified.

  Specifically, in certain embodiments, the recombinant gene marker non-human animal model described above is treated with the substance orally or intravenously, or perpetuates live bacteria by oral gavage. And the biomarker expression level of Foxp3 or IL-10 is an immunomodulatory substance in various parts of mice such as spleen, mesenteric lymph node, small intestine, large intestine, lung, pancreas, and bone marrow Expressed as a measure of ability.

  In certain embodiments, dendritic cells, T cells, T regulators, B cells, or macrophages are purified or differentiated from mice and expression of IL-10 (Foxp3 for T cell analysis) is expressed by green fluorescent protein (GFP ). In these embodiments, the candidate bacterial substance is contacted with one of the cell types described above, the GFP expression level is measured, and the immunomodulatory ability of the substance being tested will be demonstrated. On the other hand, dendritic cells or other antigen-presenting cells are cultured with the substance, the substance is washed away, and the dendritic cells are subsequently cultured with T cells, from which they generate immunomodulatory activity. Measure ability. GFP expression can be measured using a fluorescence activated cell sorter (FACS) or a microplate reader.

  In certain embodiments, the detection of at least one anti-inflammatory biolabel selected from the group consisting of IL-10, Foxp3, TGF32, perforin, and granzyme B, and selected from the group consisting of IL-17 and TNFα. T cells, antigen presenting cells, and reagents for the detection of at least one labeled inflammatory biomarker identify bacterial substances that have immunomodulatory capabilities according to the methods described herein. Can be included in the system.

  In one embodiment, the system comprises dendritic cells, T cells, T regulators, B cells, and / or macrophages purified or differentiated from mice in certain embodiments, and IL-10 (for T cell analysis). In some cases Foxp3) expression is labeled with green fluorescent protein (GFP).

  The system may be provided in the form of a kits of parts. In the kit of parts, multi-coordinate capture agents and other drugs for performing the method can be independently included in the kit. Antigen presenting cells, T cells, and reagents are included in one or more compositions, and each cell and reagent can be present in the composition with a suitable medium.

  Further components include a label-capturing drug, a microfluidic chip, specific to a labeled molecule, particularly an anti-inflammatory biological target or a molecule associated with its expression, identified by a person skilled in the art in contact with the description of this disclosure Standard products and additional components are listed.

  Details regarding suitable carrier drugs, supplemental reagents for the composition, and identification suitable for general manufacture and packaging of the kit may be recognized by those skilled in the art in connection with the description of the present disclosure.

  The vesicles, compositions, methods, and systems described herein are shown in detail in the following examples, which are described by way of illustration, and are not limited thereto.

  Specifically, the examples that follow show exemplary cell cultures, methods for contacting OMVs containing PSA (or PAS ligand) with cells and detecting the expression of biomarkers. ing. Those skilled in the art will consider the validity of the features described in detail for OMVs, PSA, Bacteroides fragilis for further vesicles, and further delivery of the substance to the PSA of OMVs or other vesicles. This disclosure will be understood.

  The following materials and methods were used for all methods and systems for the detection of immunomodulators exemplified herein.

<Bacterial strain, culture conditions, mouse>
B. fragilis strain NCTC 9343 was obtained from the American Type Culture Collection. The syngeneic PSA deletion mutant and mpi44 mutant (producing only PSA but not other polysaccharides) are described in “MJ Coyne, AO Tzianabos, BC Mallory, VJ Carey, DL Kasper and LE Comstock, (2001). ) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis, Infect. Immun. 69: 4342-4350. Bacteria can be enriched with 37 g BHI (BD # 237200), 0.5 μg / ml hemin (Sigma H5533), and 0.5 μg / ml vitamin K (Sigma V3501) in 1 L ddH ₂₀ or 1 L RPMI. (Invitrogen SKU # 11835-030) were grown in any of a special minimal medium containing 8 g glucose, 1% FBS, 0.5 μg / ml hemin, and 0.5 μg / ml vitamin K. C57BL / 6 and Balb / c strain SPF mice were purchased from Taconic Farms (Germantown, NY). TLR2 knockout mice and IL-10 knockout mice were purchased from Jackson laboratories. IL-10GFP mice were purchased from Christopher Carp Institute at Cincinnati Children's Hospital. Foxp3GFP mice were a gift from the Talal Chatila Institute at the University of California, Los Angeles.

<Isolation of EDL-rich bacterial population>
Percoll (GE Healthcare # 17-0891-01) discontinuous gradient centrifugation was performed on wild-type B. fragilis and ΔB. Fragilis (Patrick S, Reid JH. (1983) Separation of capsulate and non-capsulate Bacteriodes fragilis on a discontinuous density. gradient. J Med Microbiol. 16 (2): 239-41.) used for isolation of EDL. That is, 20%, 40%, 60%, and 80% Percoll gradients (diluted with PBS) were prepared in 14 ml test tubes (2 ml for each layer). The B. fragilis culture resuspended in PBS was then carefully added to the top of the 20% percoll layer. Further, the gradient was centrifuged at 800 g for 20 minutes at room temperature. EDL-rich bacteria were recovered from the 40% -60% interface after isolation.

<Purification and labeling of OMV>
Such methods include those previously described for the preparation of OMVs from E. coli (Amanda L. Horstman and Meta J. Kuehn. (2000) Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles J Biol Chem. 275: 12489-12496.) Was applied. That is, B.fragilis rich in EDL was grown in a special MM. OMVs were recovered from the supernatant of bacteria-free medium by centrifuging at 40,000 g for 2 hours at 4 ° C., washed twice with PBS, and then on a 0.45 μm elongated column (Millipore # 20-218). Filtered. The total protein concentration of the purified OMVs was measured by the Bradford method (Biorad # 500-0205). FITC-labeled OMVs were prepared by known methods (Nicole C. Kesty and Meta J. Keuhn. (2004) Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J Biol Chem. 279: 2069-2076). That is, OMVs were placed in staining buffer (lmg / ml FITC (Thermo Scientific # 46424), 100 mM NaCl, 50 mM Na2CO3, pH 9.2) for 1 hour at room temperature. Labeled OMVs were recovered by centrifugation at 40,000 g for 30 minutes at 4 ° C. and washed twice with PBS + 200 ml NaCl.

<Electron microscope observation of ultrathin sections of bacteria>
EDL-rich B. fragilis ultrathin sections were prepared by known methods (Patrick S, McKenna JP, O'Hagan S, Dermott E. (1996) A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles. Microb Pathog. 20 (4): 191-202.). That is, the sample was fixed overnight at 4 ° C. in a 2.5% (v / v) glutaraldehyde (Sigma, G5882) solution of cacodylate buffer, and further osmium tetroxide for 3 hours at room temperature in the dark. Fixation was performed in (1%, w / v). In any fixing treatment, ruthenium red (lmg / ml, Sigma R2751) was blended. Then, after dehydrating with a series of alcohols having a concentration gradient, the fixed sample was embedded in an epoxy resin. Ultrathin sections were cut and negatively stained with 2% uranyl acetate and lead citrate prior to TEM observation on a form bar / carbon coated copper grid (EMS # FCF200-Cu).

<Immunogold labeling of purified OMVs>
A known method was applied to this method (Patrick S, McKenna JP, O'Hagan S, Dermott E. (1996) A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles. Pathog. 20 (4): 191-202.). That is, a small drop of purified OMV was placed on a form bar / carbon coated gold grid (EMS # FF200-Au) and allowed to air dry. Immunogold labeling was performed at room temperature by floating the grid over the droplets of antibody and wash solution with the “OMVs” side of these grids facing down. Specifically, after 5 minutes in 0.12% glycine, the sample was blocked in 10% FBS for 10 minutes. Subsequently, a second antibody IgG to which 5 nm of gold was bound (provided by Dr. Paul Webster, House Ear Institute, Los Angeles) was applied to the sample, and washed with PBS again for 4 minutes 5 times. After labeling, the sample was fixed in 1% glutaraldehyde for 5 minutes, and washed with PBS for 1 minute 4 times and H ₂ O for 1 minute so that the droplets could permeate the grid. Washed well. Counterstaining was performed by placing a grid on 2% methylcellulose 3-5% uranyl acetate droplets for 10 minutes at ice temperature. Finally, the grid was removed from the staining solution using an annular wire and allowed to air dry. A sample covered with a thin film of methylcellulose was removed from the ring-shaped wire and used for transmission electron microscope observation (TEM).

<Glycoprotein analysis>
PSA purified from whole cell extracts or OMVs (derived from B. fragilis mutant mpi44) was subjected to SDS-PAGE, and the gel was then subjected to a glycoprotein staining kit (G bioscience # 786-254) to reveal the presence of PSA. ).

<Chemical experimental colitis induced by TNBS>
As this operation, a known method (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology. 15.19.1-15.19.14.) Was adopted. That is, wild type (Balb / c) male mice were treated orally with PBS, WT-OMV (5 μg), or ΔPSA-OMV (5 μg) every other day for 1 week prior to TNBS administration. Treated mice were anesthetized with isoflurane, and 2% TNBS (Sigma P2297 in 50% EtOH) was administered into the rectum via a 3.5F catheter (Instech Solomon; SIL-C35). After TNBS administration, oral administration was continued two more times, and after the last treatment, the mice were analyzed for 1-2 days.

  In the desktop experiments, before treating mice orally with PBS, WT-OMV (5 μg), or ΔPSA-OMV (5 μg) every other day for 1 week prior to TNBS administration—as described above In addition, the same mouse can be treated with TNBS. Treated mice were then anesthetized with isoflurane and 2% TNBS (Sigma P2297 in 50% EtOH) was administered to the rectum via a 3.5F catheter (Instech Solomon; SIL-C35). After TNBS administration, oral administration was continued two more times, and after the last treatment, the mice were analyzed for 1-2 days. What we predicted was that by adopting this procedure, the degree of colitis in these mice improved, improved and healed. This prediction is based on the fact that a previous study using only PSA (Round et al. 2010) prevented and reduced colitis both before and after TNBS treatment, respectively, and our results Based on the fact that in some cases the OMV action in mice is PSA concentration dependent.

<Pathological analysis of tissue>
Mouse colons were fixed in neutral 10% formalin buffer (ScyTek Laboratories CAS # 50-00-0) and processed by Pacific Pathology for H & E staining. The colitis score at each colon site was evaluated in a blinded fashion by a pathologist (Dr. Gregory Lawson, David Geffen School of Medicine, UCLA, Los Angeles). Tissue structure images were acquired using an optical microscope (Zeiss) at 20x magnification.

<Quantitative real-time PCR>
RNA was collected from mouse tissue using Trizol reagent (Invitrogen # 15596-018) or from purified cells using RNeasy mini kit (Qiagen # 74104). The iSCRIPT cDNA synthesis kit (BioRad # 170-8890) was used for cDNA conversion and IQ SYBR Green supermix (BioRad # 172-8882) was used for real-time PCR. Primers used in this test were TNF (F-5′ACG GCA TGG ATC TCA AAG AC 3 ′ (SEQ ID NO: 1)); (R-5 ′ GTG GGT GAG GAG CAC GTA GT 3 ′) (SEQ ID NO: 2) IL-17 (F-5 ′ TTA AGG TTC TCT CCT CTG AA 3 ′ (SEQ ID NO: 3)); (R-5 ′ TAG GGA GCT AAA TTA TCC AA 3 ′) (SEQ ID NO: 4); IL-10 ( F-5 'GGT TGC CAA GCC TTA TCG GA 3' (SEQ ID NO: 5)); (R-5 'ACC TGC TCC ACT GCT TGC T 3') (SEQ ID NO: 6); Foxp3 (F-5 'GCA ATA GTT CCT TCC CAG AGT TCT 3 '(SEQ ID NO: 7)); R-5' GGA TGG CCC ATC GGA TAA G 3 '(SEQ ID NO: 8); Actin (F-5' TTC GTT GCC GGT CCA CA 3 '(SEQ ID NO: 7) 9) R-5 ′ ACC AGC GCA GCG ATA TCG-3 ′ (SEQ ID NO: 10).

<Fluorescence microscopy>
In vitro differentiated BMDCs were seeded on Lab-Tek II 8-well chamber slides (Nunc # 154534) at 50,000 cells / well. FITC-labeled OMV was added to the cultured cells at 10 μg / ml. After 2 hours of culture, the cells were fixed in 4% PFA for 2 hours at room temperature. After washing with PBS for 5 minutes three times, the cells were stained with 1 μg / ml of WGA (Invitrogen W849) tetramethylrhodamine complex at 4 ° C. for 1 hour. Then, prolong gold anti-fading agent (P36930) was applied to the thoroughly washed cells after membrane staining. Fluorescence images were acquired using an LSM 510 microscope and an oil immersion Plan-Neofluar 63x / 1.25.

<Flow cytometry method and staining method>
BMDC derived from OMV acquisition operation or activity operation was collected and blocked in 5% mouse serum at ice temperature for 30 minutes. After block treatment, stain with anti-CD11c-APC, anti-MHCII-FITC, or anti-CD86-PE (ebioscience) for 30 minutes at ice temperature and FACS buffer at 4 ° C before flow cytometry Washed twice with liquid (HBSS (without Ca2 + / Mg2 +), 1% FBS, 2 mM EDTA, 10 mM HEPES). Similarly, anti-CD4-APC /, except that cells derived from BMDC-T cell co-cultures in vitro were blocked and restimulated with PMA / ionomycin for 4-4.5 hours prior to harvesting. Staining was similarly performed using anti-CD25-PE. All flow cytometry was performed using BD FACSCalibur and the results were analyzed using FlowJo.

<Co-culture of BMDC-T cells in vitro>
Bone marrow was collected from different mouse strains and as already known (Mazmanian, Liu, Tzianabos, and Kasper (2005) An Immunomodulatory Molecule of Symbiotic Bacteria Directs Maturation of the Host Immune System. Cell 122: 1 107-118) , Differentiated in vitro for 8 days in the presence of 20 ng / ml GM-CSF (Miltenyi Biotec # 9517571) (cell purity> 90%). CD4 + splenic T cells were isolated by magnetic bead purification (Miltenyi Biotec # 130-090-860) (cell purity> 95%). BMDCs targeted with OMV (10 μl / ml OMVs, 100,000 cells / ml, 12-24 hours) were washed with HBSS and anti-CD3 was added to 0.01 μl / ml (day 0, FIG. 3C 3D, 3E, 3F, FIG. 4A), 2 ng / ml TGFb added (0 days, FIGS. 3C, D and F, FIG. 4A) and 5 ng / ml II-2 added (1 day and 3 days, all in vitro DC-T cell co-culture method) was cultured together with CD4 + T cells (1,000,000 cells / ml) in a round bottom 96-well plate. After 4 days in culture, supernatants were collected for ELISA (ebioscience # 88-7104-77) or cells were collected for staining or flow cytometry.

<Inhibition procedure in vitro>
BMDC (labeled with WT-OMV or APSA-OMV) -CD4 + CD25 + cells purified from T cell co-cultures were used as the source of Treg (Miltenyi Biotec, # 130-091-041). CD4-deficient mouse spleens treated with mitomycin C (Sigma M4278) were used as APCs (100,000 cells / ml). CD4 + CD25-T cells purified directly from mouse spleen are targeted with CFSE for 10 minutes at 37 ° C., followed by a first wash with PBS, a second wash with medium, and killer cells. Used immediately as (Teff) (500,000 cells / ml). This procedure was performed in a 200 μl volume 96-well round bottom plate supplemented with 5 μg / ml anti-CD3 (ebioscience # 16-0031-86). Teff: Treg ratio was titrated and cells were harvested for FACS analysis after 2-3 days of culture.

<Statistical analysis>
Student T test and one-way ANOVA were employed for paired comparisons and comparisons of two or more groups, respectively. To identify groups showing statistical significance, significant differences between groups by ANOVA were analyzed by using the Newman-Keuls test as a post-hoc test. All error bars indicate SEM (error range). NS: no significant difference, * p <0.05; *** p <0.01; *** p <0.001.

(Test Example 1)
Immunoadaptable capsular polysaccharide PSA is actively loaded into B. fragilis OMVs.

  B. fragilis EDL-rich ultrathin sections were prepared as described in Materials and Methods and photographed with a transmission electron microscope.

  The results shown in FIG. 1A indicate that OMVs are abundantly produced by the bacteria and exit outward from the bacterial outer membrane (FIG. 1A, higher magnification). In the prior art, it has been shown that deletion of PSA abolishes the immunomodulatory ability of B. fragilis (Mazmanian, Liu, Tzianabos, and Kasper (2005) An Immunomodulatory Molecule of Symbiotic Bacteria Directs Maturation of the Host Immune System. Cell 122: 1 107-118.) (Mazmanian, Round, and Kasper (2008) A microbial symbiosis factor prevents intestinal inflammatory disease. Nature. 453 (7195) 620-625.). Electron micrographs of PSA mutants (B. fragilisΔPSA) show no defects in OMV synthesis and show that the size, shape and abundance of produced OMVs are indistinguishable from wild type bacteria (FIGS. 1A and 5). Specifically, the results shown in FIG. 1A indicate that vesicles are actively emerging from the bacterial surface.

  To determine whether PSA is associated with B. fragilis OMVs, purified vesicles from wild-type and ΔPSA bacteria were subjected to immunoblot analysis as described in the Materials and Methods section. did.

  The results shown in FIG. 1B indicate that, unlike OMVs derived from B. fragilis ΔPSA, vesicles derived from wild type show immunological activity against PSA. B. fragilis produces at least eight different capsular polysaccharides that cover the bacterial surface, which are referred to as PSA, PSB, PSC, PSD, PSE, PSF, PSG, and PSH. PSB is detected in the vesicle preparation while PSG is absent. This demonstrates the selectivity of the given polysaccharide for inclusion in OMVs (FIG. 1B). Thus, the results in FIG. 1B indicate that while PSA and PSB are associated with vesicles, PSG is only observed on the bacterial surface. Capsular polysaccharide deletion mutants ensure selectivity for each antiserum.

  The results from immunoblot analysis were supported by immunogold labeling studies performed as described in the Materials and Methods section. The result of immunogold labeling of purified vesicles is shown in FIG. 1C, which shows that PSA is physically associated with OMVs and that most of the OMVs from wild-type B. fragilis are positively stained by PSA. Is supported (FIG. 6). To verify that the absence of PSA does not change the molecular composition of OMVs, proteomic analysis was performed by mass spectrometry, which greatly increased the protein composition between vesicles derived from wild-type bacteria or PSA-mutant bacteria. It was found that there was no quantitative or qualitative difference (FIG. 7).

  PSA is a heterogeneous polymer compound having repeating units. Chromatographic separation of PSA recovered from whole cell extracts was performed, immunoblot analysis using capsular polysaccharide anti-PSA prepared from whole cells was performed, and OMVs were purified as indicated in Materials and Methods .

  The relevant results shown in FIG. 1D surprisingly indicate that only a low molecular weight species, L-PSA, is associated with OMVs, indicating specific uptake of PSA into vesicles. Specifically, the results of FIG. 1D show that only low molecular weight PSA (L-PSA) is taken up by vesicles, whereas high molecular weight (H-PSA) species remain associated with the bacterial outer membrane. It is shown that.

  Based on the above results, immunoregulatory capsular polysaccharide PSA actively enters into OMVs of B. fragilis.

(Test Example 2)
OMVs protect animals from experimental colitis and intestinal inflammation in a PSA concentration dependent manner.

  To investigate whether OMVs can improve the clinical symptoms of the disease, mice were administered by oral gavage with OMVs during TNBS (2,4,6-trinitrobenzenesulfonic acid) -induced colitis. Treated.

  The results shown in FIG. 2A show that control animals rapidly lost weight after rectal administration of TNBS (FIG. 2A, TNBS + PBS), vehicle-treated mice (FIG. 2A, ETOH + PBS). ) And did not recover. Of note, TNBS mice given OMVs orally were especially prevented from losing weight (FIG. 2A, TNBS + WT-OMV). Most importantly, when OMVs derived from B. fragilisΔPSA are administered, weight loss becomes indistinguishable from TNBS animals (FIG. 2A, TNBS + ΔPSA-OMV), which involves PSA in preventing debilitating disease Prove that you are.

  Our attempt to detect intact vesicles in the colon after gavage was confused by observation of host-derived vesicles, and as already reported (no experimental data), aseptic (fungal It was also confusing in mice.

  Because colon length reduction is characteristic of TNBS colitis, colon length measurements were made in the untreated intestine immediately after removal from the cecum to the rectum, and the vehicle treated group (EtOH) and TNBS group ( Quantification (graph) of the length of n = 4 animals / group) was performed. Specifically, the measurement was performed after death (4 days after disease induction).

  The results shown in FIG. 2B show normal bowel length in animals treated with PSA-containing vesicles and not in animals treated with OMVs without PSA (FIG. 2B).

  The results of experimental colitis caused severe pathological changes in the intestinal structure. Thus, hematoxylin and eosin (H & E) stained intestinal sites representing each treatment group were provided as shown in FIG. 2C. The image in FIG. 2C shows that, in histological analysis of colon tissue, significant disease was observed in TNBS-treated animals improved by oral administration of PSA-containing vesicles.

  The results in FIG. 2C were confirmed by colitis scores in animals that had TNBS colitis in local lesions and assigned by a blinded pathologist through the intestine similar to those observed in Crohn's disease. In order to quantitatively assess disease, clinical symptoms were evaluated using a standard scoring system (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology. 15.19.1-15.19.14.) Assessed by a blinded pathologist. The results shown in FIG. 2D showed that WT-OMVS significantly reduced disease in most animals, although all animals treated with TNBS and ΔPSA-OMV were severely affected. Animals treated with OMVs from wild-type bacteria had few pathologically significant diseases, and when lesions were observed, the lesions were small compared to animals given ΔPSA-OMV, almost The normal tissue structure was maintained.

  The above results demonstrate that PSA is required for disease prevention activity of OMVs.

(Test Example 3)
PSA containing OMVs suppresses TNFα / IL-17 expression and enhances IL-10 expression.

  Production of standard inflammatory and anti-inflammatory cytokines associated with colitis was measured in mice treated as exemplified in Test Example 2. Specifically, cytokine transcription analysis was performed by qRT-PCR of RNA recovered from whole intestine or purified CD4 + T cells derived from mesenteric lymph nodes.

  Related results representing three independent studies are reported in FIG. 2E (whole intestine) and FIG. 2F (purified CD4 + T cells from mesenteric lymph nodes). These results indicate that inflammatory biomarker / cytokine tumor necrosis factor (TNF-α) and IL-17A transcription levels are elevated in TNBS-treated animals but are reduced by OMV administration depending on PSA concentration. It is shown (FIG. 2E). Consistent with protection from pathology, OMVs elicited increased production of anti-inflammatory biomarker / cytokine IL-10 levels compared to animals given orally ΔPSA-OMV (FIG. 2F). Cytokine analysis produced by purified CD4 + T cells derived from mesenteric lymph nodes (MLNs) has ensured that IL-10 is indeed produced by T cells in response to PSA (Fig. 2F). Th17 cell infiltration required for disease was significantly reduced by OMVs, resulting in the level of TNF-α, a potent inflammatory marker (FIG. 2F). We conclude that entry of PSA into B. fragilis OMVs protects animals from the pathological and immunological symptoms of experimental colitis.

(Test Example 4)
PSA containing OMVs from B. fragilis contains a dendritic cell response

  Dendritic cells (DCs) protrude into the intestinal lumen, prey on intestinal particles, and then migrate to MLNs to initiate a T cell response. Indeed, PSA orally administered to animals is associated with CD11c + DCs in MLN. Therefore, Applicants sought to test whether OMVs containing PSA could also be incorporated into DCs.

  The results show that bone marrow-derived DCs rapidly internalize OMVS, depending on the actin concentration, because treatment of cells with cytochalasin D inhibits vesicle uptake (FIG. 3A). And FIG. 8). Intracellular localization was confirmed by confocal microscopy (FIG. 9).

  PSA-mediated induction of T cell activation markers in DCs was also examined. The results shown in FIG. 3B show that the expression of T cell activation markers (MHCII, CD86) is equally increased following internalization of both WT-OMVS and ΔPSA-OMVs (see FIG. 3B and FIG. 10). reference). Increased expression of MHC and costimulatory molecules indicates that PSA containing OMVs from B. fragilis affects T cell responses.

(Test Example 5)
PSA containing OMVs induces IL-10 expression in CD4 + T cells by DCs IL-10.

  Considering the protective function of OMVs-containing PSA in colitis shown by the experiments exemplified in Test Examples 1 to 3, the biological effect of OMVs in inducing the response of suppressor T cells was tested. Specifically, the ability of OMVs to promote Treg cell development was tested as a population of various regulatory T cells (Tregs) known to inhibit colitis.

  The results shown in FIG. 3C indicate that WT-OMVS induces IL-10 expression from DC-T cell co-culture in vitro, whereas under these conditions only IL-10 It is not produced from DCs. Although increases were detected in media control, vesicles purified from B. fragilisΔPSA induced significantly less IL-10 than WT-OMVS. Production of IL-10 from DCs is known to support CD4 + IL-10 + T cell development in vivo and in vitro. Therefore, an ELISA analysis similar to that shown in FIG. 3C was performed to include DCs differentiated from IL-10 − / − animals.

  The results shown in FIG. 3D (left side) show that IL-10 production is significantly reduced in DC-T cell co-culture when IL-10-/-DCs are treated with wild-type OMVs. Suggests the expression of IL-10 by DCs and requires that IL-10 be induced from CD4 + T cells in a paracrine manner.

(Test Example 6)
PSA containing OMVs directs DCs to direct Foxp3 Treg development and / or expansion.

  Microbial ligands are sensed by several classes of pattern recognition receptors, and PSA has been shown to signal through Toll-like receptor 2 (TLR2) to induce Th1 cytokine production. A series of experiments were therefore conducted to test whether TLR2 is required for induction of IL-10 by wild-type OMVs (including PSA), and recent reports show that Treg function and IL- 10 expression has been shown to be affected by TLR2.

  The results in FIG. 3D (right side) show that the absence of TLR2 (TLR2 − / − animal-derived DC) is completely IL-10 produced in response to OMVs compared to wild-type DCs. It is to suppress to. Both DCs responded similarly to superantigen (SEA) stimulation, showed specific deficiencies in PSA sensing, and did not show a general deficiency of T cell activity by TLR2-/-DCs (Fig. 3D). CD4 + CD25 + T cells expressing the transcription factor Foxp3 are an important Treg subset. What has been shown in recent studies is that CD4 + CD25 + FOXP3 + Tregs express IL-10 and IL-10 production from Tregs is required to prevent intestinal inflammation.

  To measure the source of IL-10 induced by PSA in OMVs, CD4 + CD25 + T cells and CD4 + CD25-T cells were purified after co-culture with DCs to express IL-10 and Foxp3 Was measured by qRT-PCR.

  The results described in FIG. 3E indicate that PSA-OMV significantly induces IL-10 expression but not from CD4 + CD25- T cells, particularly in the CD4 + CD25 + Treg population. That is. OMVs purified from B. fragilisΔPSA are unable to promote IL-10 production from T cell populations to the same extent as control media. In a PSA-dependent manner, Foxp3 expression was also significantly increased by OMVs in CD4 + CD25 + T cells (FIG. 3E).

  Co-culture was set up similar to the experiment shown in FIGS. 3C-E, but CD4 + T cells from Foxp3-GFP mice were used. After 4 days in culture with DC targeted with OMV, the cells were stained with anti-CD4 and Foxp3 detected by GFP expression using FC.

Representative results of two independent tests are shown in FIG. 3F.
Consistent with increased Foxp3 transcription in CD4 + T cells, the ratio of CD4 + Foxp3 + T cells increased in response to WT-OMV treatment rather than PSA-OMV treatment (FIG. 3F). Collectively, OMVs containing PSA direct DCs directly to Treg development and / or expansion in a completely in vitro culture system.

(Test Example 7)
PSA-containing OMVs induce an IL-10-mediated Foxp3 Treg inhibitory effect.

  The use of Tregs as cell therapy has been proposed for IBD, autoimmunity, and allergies. IL-4 expression by CD4 + T cell subsets was examined after 4 days of co-culture with DCs treated with OMVs. Splenic CD4 + T cells were purified from IL-10-GFP mice, co-cultured and stained with anti-CD4 and anti-CD25, and IL-10 expression was measured by GFP expression.

  The results in FIG. 4A of the associated OMV treatment of purified DCs and T cells indicate that PSA promotes specific production of IL-10 from CD4 + CD25 + Treg.

  Based on this finding, the ability of PSA was examined and killer cells in vitro with purified CD4 + CD25 + T cells after co-culture with DCs treated with CD4 + CD25, medium (control), OMVs, ΔPSA-OMVs And to promote the ability to suppress ex vivo suppression. CD4 + CD25-killer cells (effector cells, Teff) were purified from the spleen of wild-type mice, identified with the intracellular dye CFSE (carboxysuccinimidyl ester), cultured with Tregs, and stimulated with anti-CD3 for 3 days It was done. Cell proliferation was measured by CFSE diluted FC.

  Results shown in FIGS. 4B and 4C show that Tregs recovered from the situation with DC treated with WT-OMV was significantly enhanced compared to CD4 + CD25 + T cells responding to ΔPSA-OMVS Is displayed.

  Thus, the specific absence of PSA from OMVs, including many other microbial ligands (LPS, lipoproteins, peptidoglycans, etc.) eliminates the ability of B. fragilis vesicles to induce functional Tregs.

(Test Example 8)
PSA induces various biomarker combinations in Foxp3 Treg.

  Foxp3-GFP mice were treated orally with PSA purified every other day for 6 days. MLNs were extracted and CD4 + Foxp3 + T cells or CD4 + Foxp3-T cells were purified by FACS based on ± GFP expression (purity> 99%). RNA was extracted and used for q-PCR.

  The results shown in FIG. 11 indicate that PSA induces multiple anti-inflammatory genes such as IL-10, TGF-β, perforin, and granzyme A, and inhibits TNFα and IL17 (particularly IL17A). It has been demonstrated to be linked.

  The above results provide a molecular mechanism of B. fragilis probiotic activity and demonstrate an influential example for microbial ligands associated with immune receptor transmission to regulatory T cells doing. Specifically, the above results indicate that PSA is sent to the host by outer membrane vesicles (OMVs) that target bacterial molecules to the host cell, the secretory structure PSA. PSA-containing OMVS is internalized by dendritic cells of the host immune system. After uptake of OMVS, PSA directs dendritic cells to induce differentiation of regulatory T cells (Treg) that express Foxp3 and the anti-inflammatory cytokine interleukin-10 (IL-10). Treg development by OMVS requires the expression of toll-like receptor 2 (TLR2) and IL-10 production by dendritic cells. Surprisingly, in a PSA-dependent manner, purified OMVS induces differentiation of functional Tregs in vitro with potent inhibitory activity. Treating animals with OMVs containing PSA prevents experimental colitis and suppresses inflammatory cytokine responses in the intestine. These findings demonstrate that the symbiotic bacteria provide a beneficial microbial factor through vesicular secretion, a process designed into a novel approach for the transmission of probiotic therapy for IBD.

  Specifically, the above results indicate that Tregs induced by OMVs containing PSA are functionally suppressed and inhibit the activity of T cells in culture. This is the first demonstration of in vitro Treg development by microbial ligands and provides a proof of principle for the widely speculated idea that innate immune receptor signals regulate Treg cells. Because current therapies for IBD are ineffective or have significant side effects, probiotics are promising new treatment options by utilizing advanced and innovative mechanisms for immune regulation It corresponds to. Considering the suppression of immune responses in multiple tissues, our ingenious PSA in vitro Treg development is a novel cell therapy for many inflammatory diseases such as autoimmunity, asthma and allergies Suggests an interesting possibility.

  In order to identify molecules made by microorganisms that mediate immune regulation, the same procedure is followed until the bacterial product fraction has been purified to obtain pure compounds with similar results when whole bacteria are used. To be done. The above approach can also be used to screen a collection of microbial mutations of interest to identify immunoregulatory molecules that are missing in each clonal strain having this activity.

  Our previous study has shown that PSA alone treats and prevents colitis in mice (Round et al. 2010). Thus, our results show that wild-type OMV containing PSA prevents colitis in mice and that this effect using OMV is dependent on our proposed PSA. It is conceivable that wild OMV is effective in treating colitis in the same TNBS mouse model and has beneficial or ameliorating activity for humans with IBD.

  It should be noted here that, although markers for predicting the onset of inflammatory diseases (IBD, etc.) are not known, these once-predicted markers have been identified as substances / compositions as described above to prevent the onset of the disease. By thing / OMVs is meant that things / humans at risk of disease progression are treated as well.

  In another embodiment, OMVs (including PSA) can be modified to prevent the expression of a given enzyme or to reduce the expression of a given enzyme. Aggregation activity and enzyme activity (eg, hydrolytic enzymes such as alkaline and acid phosphatases, esterases, lipases, phosphohydrolases, glucosaminidases) are reduced using molecular or physical methods known to those skilled in the art. Such modified OMVs can then be used to treat or prevent inflammation or inflammatory diseases.

  The above examples are provided to provide those skilled in the art with a complete disclosure and description for using and implementing embodiments of the disclosed vesicles, systems, and methods to the art. It is not intended to be limiting as to the scope of the disclosure. Variations of the above-described manner apparent to those skilled in the art to practice the present disclosure are intended to be within the scope of the following claims. All patent publications and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which this disclosure pertains. All references described in this disclosure are incorporated by reference to the same extent as each reference is individually incorporated by reference in its entirety.

  The entire disclosure of the technical background, abstract, detailed description, and each description (including patent publications, patent application publications, academic papers, abstracts, experimental manuals, books, or other disclosures) described in the examples is by reference. Incorporated herein by reference.

  It should be understood that the present disclosure is not limited to a particular composition or biological system, and can, of course, vary. Also, the terminology used herein should be understood as being for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” refer to “multiple” unless the content clearly dictates otherwise. Includes. The term “plurality” encompasses two or more objects, unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

  Any methods and materials similar or equivalent to those described herein can be used to test specific examples of suitable materials and methods.

  A number of disclosed embodiments have been described. Nevertheless, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

  Although the present invention has been described in connection with a preferred embodiment, it should be understood that improvements and modifications can be utilized without departing from the principles and scope of the present invention, as will be readily appreciated by those skilled in the art. Accordingly, such modifications may be practiced within the scope of the following claims.

<References>
1.Xavier, RJ & Podolsky, DK Unravelling the pathogenesis of inflammatory bowel disease.Nature 448, 427-434 (2007).
2. Mazmanian, SK, Round, JL & Kasper, DL A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620-625 (2008).
3. Braun, J. & Wei, B. Body traffic: ecology, genetics, and immunity in inflammatory bowel disease. Annu Rev Pathol 2, 401-429 (2007).
4. Round, JL & Mazmanian, SK The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol (2009).
5. Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology. 15.19.1-15.19.14.
6. Mazmanian, Liu, Tzianabos, and Kasper (2005) An Immunomodulatory Molecule of Symbiotic Bacteria Directs Maturation of the Host Immune System. Cell 122: 1 107-118
7. MJ. Coyne, AO Tzianabos, BC Mallory, VJ Carey, DL Kasper and LE Comstock, (2001) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis, Infect. Immun. 69: 4342-4350.
8. Patrick S, Reid JH. (1983) Separation of capsulate and non-capsulate Bacteriodes fragilis on a discontinuous density gradient. J Med Microbiol. 16 (2): 239-41
9. Amanda L. Horstman and Meta J. Kuehn. (2000) Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem. 275: 12489-12496.
10. Nicole C. Kesty and Meta J. Keuhn. (2004) Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J Biol Chem. 279: 2069-2076.
11. Patrick S, McKenna JP, O'Hagan S, Dermott E. (1996) A comparison of the
haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles.Microb Pathog. 20 (4): 191-202.
12. Round et al. (2010) Inducible Foxp3 + regulatory T cell development by a commensal bacterium of the intestinal microbiota. PNAS, 107 (27). 12204-12209.

Claims (4)

  A medicinal composition for use as a drug, comprising an outer membrane vesicle formed of a lipid membrane surrounding an aqueous environment and containing Polysaccharide A (PSA), wherein the lipid membrane is obtained from a cell membrane of Bacteroides fragilis.   The medicinal composition according to claim 1, further comprising an excipient.   The medicinal composition according to claim 1 or 2, wherein the PSA is low molecular weight PSA (L-PSA).   The medicinal composition according to any one of claims 1 to 3, further comprising one or more known drugs useful for the treatment of inflammatory bowel disease.